Mandibular prognathism (Online Mendelian Inheritance in Man number 176700), skeletal Class III malocclusion in orthodontics, is a multifactorial phenotype. Some linkage analyses and supplementary association studies for mandibular prognathism have been carried out in various races. From the results of linkage analysis, Yamaguchi et al suggested that chromosomes 1p36, 6q25, and 19p13.2 were associated with mandibular prognathism in Japanese and Korean families. Association studies examining the putative susceptibility locus close to 1p36 suggested that Matrilin-1 (cartilage matrix protein) and EPB41 (erythrocyte membrane protein band 4.1) were candidate genes of the phenotype. In contrast, another linkage analysis of Brazilian families found that 1p36, 6q25, and 19p13.2 were not associated with the phenotype. Furthermore, Frazier-Bowers et al suggested from results of linkage analysis of Colombian Hispanic families that 5 susceptibility loci—1p22.1, 3q26.2, 11q22, 12q13.13, and 12q23—were related to the phenotype. An association study suggested that MYO1H (myosin 1H) located on 12q24.11, close to 12q23, was a candidate gene of the phenotype. Two more linkage analyses examining the susceptibility loci of the phenotype have been reported. Genome-wide association study (GWAS) has been proposed as an alternative strategy for linkage analysis. There have been no reports on GWAS of mandibular prognathism. The linkage analyses of Yamaguchi et al and Frazier-Bowers et al suggested that susceptibility loci of mandibular prognathism were on chromosome 1. We therefore show the results of an association study at chromosome 1 for mandibular prognathism.

The preliminary association study of Japanese patients suggested that 2 loci (1q32.2 and 1p22.3) were susceptibility regions of mandibular prognathism and that PLXNA2 and SSX2IP were candidate genes. 1p32.2 was a novel locus, and 1p22.3 corroborated the previous suggestive linkage analysis results. After our preliminary study on chromosome 1, more linkage or association analyses on susceptibility loci of the disease have been reported in the last 2 years. The objective of this study was to extend the data gained from our previous association study of mandibular prognathism on chromosome 1 to the whole GWAS, using the same cohort and methodology as previously described.

Material and methods

The study protocol was approved by the ethics committees of the School of Dentistry and Pharmaceutical Science of Hokkaido University and the School of Medicine of Tokai University. All subjects gave written informed consent for participation in the study. Two hundred forty subjects with mandibular prognathisms and 360 healthy controls of Japanese descent were recruited as previously described. Inclusion and exclusion criteria for subjects diagnosed with mandibular prognathism were also the same as described previously. There were no biologic relationships among the subjects.

Genomic DNA was isolated from whole blood cells using the QIAamp DNA Blood Maxi Kit (QIAGEN, Hilden, Germany). Microsatellite genotyping was performed as described in our previous study using a pooled DNA method. The allele frequencies for pooled DNA typing were estimated by the height of each peak. To reduce the number of pseudo-positives resulting from type I errors, 2 steps of screening were performed to sequentially replicate the results in each pooled sample. Pooled DNA was screened using a dense panel of 23,465 microsatellites covering the whole genome in the first screening. Microsatellites showing statistical significance ( P <0.05) in the first screening were subjected to the second screening ( Fig ).

Flow chart of the first and second screenings and individual genotyping. Independent samples were used for the first and second screenings. In individual genotyping, we removed 18 microsatellites from 36 positive microsatellites because the 18 microsatellites deviated from the Hardy-Weinberg equilibrium or coded gene deserts. Individual genotyping was performed in the full sample of 240 subjects and 360 controls.

After the first and second typings using pooled DNA, microsatellite markers that showed a significant association with mandibular prognathism in both the first and second typings were further screened using individual-subject DNA samples from the pooled DNA set (240 subjects and 360 controls). Amplification of each microsatellite from each DNA sample was conducted using polymerase chain reaction to verify significant associations ( Fig ). The polymerase chain reaction conditions and method for analyzing polymerase chain reaction products were the same as previously described. The allele frequencies for individual genotyping were estimated by the height of each peak.